The present invention relates to materials and methods for prevention, treatment and diagnosing of infections caused by Helicobacter pylori. More specifically the present invention relates to polypeptides and antibodies useful in vaccines for the treatment and prevention of pathologic infections caused by Helicobacter pylori strains. The present invention specifically relates to a bacterial blood group antigen binding adhesin (BAB-adhesin). The present invention further relates to polynucleotides useful for the recombinant production of said polypeptides and for use in immunisation therapies. In addition, it relates to polypeptides, antibodies, and polynucleotides used for the detection of said bacteria.
The present invention further relates to new immunoglobulins, which exhibit specific activity to a blood group binding adhesin, expressed by Helicobacter pylori, methods for the production of said immunoglobulins, their isolation and use. The present invention further relates to the treatment and prevention of H. pylori induced infections in the gastrointestinal tract.
Helicobacter pylori is a causative agent for acid peptic disease and the presence of this organism is highly correlated to the development of gastric adenocarcinoma. Bacterial adherence to the human gastric epithelial lining was recently shown to be mediated by fucosylated blood group antigens.
Recent research has focused on the role of Helicobacter pylori in the development of ulcers in the gastric mucosa. Recent findings show a strong connection between H. pylori and chronic, active gastritis and gastric ulcers. Furthermore, there appears to be a strong correlation between ventricular cancer and gastric ulcers. Traditional treatment of gastric ulcers has involved gastric resection, the administration of bismuth compositions, the administration of H2-blockers and the administration of pH-buffering agents, to mention a few examples.
More recently, various forms of treatment have been supplemented with the administration of antibiotics. One problem with presently known treatments is the risk for treatment failure. Furthermore, not only do microbes develop antibiotic resistance, the antibiotics administered often upset the natural balance of benign microbes, colonising the intestinal tract. This leads to diarrhoea and other signs of intestinal discomfort, in addition to destabilising the benign flora in the intestines. Other treatments, e.g. H2-blockers often require life-long medication to prevent the recurrence of disease.
The foregoing, together with the fact that H. pylori is very widely spread among humansxe2x80x94according to a conservative estimate approximately 60% of all adult humans in the industrialised countries are infectedxe2x80x94makes the diagnosing, prevention and treatment of H. pylori infections an urgent task.
Further, the fact that developing countries frequently lack the resources for conventional treatment of gastric ulcers further underlines the importance of finding new ways of treatment and prevention of H. pylori induced infections. It is obvious, for many reasons, that disease prevention with vaccines is a preferable mode. A vaccine would provide an easily administered and economical prophylactic regimen against H. pylori infections. An effective vaccine against H. pylori is nevertheless presently lacking.
H. pylori colonises the human gastric mucosa, in an equilibrium between adherence to the epithelial surface mucous cells and the mucous layer lining the gastric epithelium. Once infected, bacteria seems to colonise for a lifetime. Attachment to the epithelial lining protects the bacteria from the anti-microbial effects of the acidic gastric juice of the stomach lumen, as well as from physical forces such as peristalsis. For survival in this hostile ecological niche, H. pylori has developed a battery of virulence factors; such as production of the enzyme urease that buffers the micro-environment around the bacteria and the polar flagellae to ensure high motility, a prerequisite in an ecological niche where the turnover of the mucous layer is in the range of hours. A subset of H. pylori strains produces the vacuolating cytotoxin, VacA, and the cytotoxin associated antigen CagA.
Attachment is essential for colonisation of the epithelial lining and bacteria express surface associated adhesion molecules that recognise specific carbohydrate or protein receptors on the cell surfaces or mucous lining. The specificity in this interaction in combination with the genetically regulated receptor distribution results in a restricted range of cell lineages and tissues available for colonisation. Several putative receptor structures have been described for H. pylori, such as the hemagglutinin-sialic acid, sulphated glycoconjugates and sulphatides. Recently, the fucosylated blood group antigens H-1 and Lewisb were described (Borxc3xa9n et al., Science, 262, 18921993), mediating specific adherence of H. pylori to human and rhesus monkey gastric surface mucous cells in situ. The H-1 and Lewisb antigens are part of the blood group antigens that define blood group O in the ABO system.
Surface-exposed proteins are often constituents of the outer membrane. The outer membrane has a structural role and acts as a selective barrier, determining what enters the cell and what molecules are secreted. One class of outer membrane proteins are called porins, and create hydrophilic pores through the outer membrane where specific metabolites, such as sugar molecules, can cross. Recently the finding of a number of outer membrane proteins in H. pylori, was reported, which proteins were suggested to constitute a family of porin proteins.
The BAB adhesin has previously been identified and shown to be localised on the bacterial surface of H. pylori (SE 9602287-6). The blood group binding activity was shown to be pH dependent and the present inventors present evidence that the binding affinity to the Lewisb receptor reveals a high equilibrium constant. For the purification of the BAB adhesin, a crosslinker-labelled receptor conjugate was used in order to mediate specific transfer of biotin to the adhesins on the bacterial surface. Thereafter the biotin-labelled adhesin could be extracted by streptavidin coated magnetic beads. Determination of the amino terminal amino acid sequence of the purified BAB adhesin exhibit homologies to outer membrane proteins of H. pylori porins.
Intensive research has been directed to the immunological treatment and prevention of H. pylori induced infections. EP 0 484 148 (Ando and Nakamura) describes a method for treating and/or preventing upper gastrointestinal disease in mammals, said method comprising orally administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising anti-Helicobacter pylori polyclonal immunoglobulins and a pharmaceutically acceptable carrier. Said description further dwells on the combination of said treatment in combination with the administration of antibiotics. As the method of producing said polyclonal antibodies, EP 0 484 148 describes the isolation and purification of anti-H. pylori immunoglobulins from the sera and milk of mammals. H. pylori itself was not found in the stomachs of cows, goats, sheep, swine or horses, according to EP 0 484 148, but it was assumed that these animal species have colonizing microorganisms with antigenic determinants similar to those of H. pylori because they have immunoglobulins which cross-react to strains of H. pylori found in humans. Preferably, according to EP 0 484 148, large mammals, e.g. pregnant cows, are immunized with whole cells of H. pylori and the immunoglobulins subsequently extracted from the milk or colostrum. In the immunization experiments, NCTC Strain 11362 and clinical isolate H. pylori No. 153 were used to trigger the production of immunoglobulins. On the other hand, NCTC Strain 11637 was used for analysing purposes. Immunization is claimed to yield an anti-H. pylori titer in the milk of such magnitude, that daily doses of 0.01-0.1 g/day immunoglobulin composition, are sufficient for successful therapy. The claimed interval of 0.01-0.1 g/day is however not supported by the experiments presented by Ando and Nakamura and so low doses have hitherto not proven efficient in clinical tests. The doses actually used in example 5 and 7 are in the order of magnitude of 1 g/day, i.e. 10-fold the upper limit of the given interval. Furthermore, it is very unlikely, that unspecific immunoglobulin mixtures as those manufactured by Ando and Nakamura, would be effective in claimed doses as similar doses are ineffective against other gastrointestinal pathogens. The simultaneous administration of antibiotics, extensively discussed in the description, underlines the insufficiency of the disclosed immunoglobulins.
EP 0 469 359 (Cordle and Schaller) likewise describes the immunization of mammals, preferably pregnant cows, with formalin killed H. pylori bacteria (ATCC Strain 26695). Anti-H. pylori polyclonal antibodies were isolated and purified from the milk and finally fed to piglets, in amounts of about 0.5 g immunoglobulins, three times daily. The results were assessed by determination of the number of biopsy specimens, which were positive for Gram-negative bacteria after the trial. Gram-negative bacteria was found in 78% of the piglets fed a non-immune nutrient but only (Sic!) in 35% of the piglets fed a nutrient containing so called specific anti-H. pylori antibodies.
Anti-H. pylori polyclonal antibodies, effective to cause aggregation of H. pylori, have thus been administered orally as a regimen in the treatment and prevention of H. pylori induced infections in the gastrointestinal tract. Nevertheless, as also noted in EP 0 484 148 A1, it is still not clear, how many antigenic determinants are present on the surface of H. pylori. The occurrence of a wide variety of H. pylori strains, makes questionable the practical efficiency of any polyclonal immunological therapy according to the state of the art. Immunization using whole bacteria will always trigger a highly polyclonal immunresponse with a low level of antibodies against a given antigenic determinant. This is underlined e.g. by the results presented by Cordle and Schaller, where, although the number of Helicobacter positive biopsies were reduced, complete cure was not obtained through the treatment according to their invention.
It is notable, that the dose of immunoglobulin needed for oral prophylaxis or therapy has not yet been clearly defined. In a normal human adult, approximately 5 g IgA is produced and secreted at mucosal surfaces each day. Obviously, doses of this magnitude are economically and practically unfeasible for large-scale therapy or prophylaxis. In studies on the effect of oral immunoglobulin on rotavirus infection, daily doses in the interval of 600 to 9000 mg have been tried in clinical tests. Successful intervention has also been reported when treating H. pylori and cryptosporidial infections with daily administrations of 3 to 15 g immunoglobulin from immunized cows (Hammarstrxc3x6m et al., Immunol Rev, 139 (1994) 43-70). Generally speaking, all studies hitherto point to the necessity of using high doses of immunoglobulins when trying to combat an ongoing infection. The need for more specific immunoglobuline preparations, allowing the use of smaller doses, is thus an urgent one.
To maximize the potency of an immunological regimen for the treatment and prevention of H. pylori, it is of great importance to find a specific conserved antigenic determinant, which plays a central role for the pathogenicity of H. pylori. Using such an antigenic determinant would make it possible to produce highly specific and therapeutically efficient novel polyclonal and/or monoclonal immunoglobulin preparations.
The above problem of providing specific, cost-efficient and therapeutically superior immunoglobulin preparations for the treatment and prevention of H. pylori has now been solved through the composition and methods according to the attached patent claims. The present inventors have now surprisingly shown, that highly specific and therapeutically efficient polyclonal and/or monoclonal immunoglobulin preparations can be provided through the immunization of an animal with an adhesin protein, specific for H. pylori. The invention will now be described in closer detail with reference to the attached, non-limiting figures and examples.
One objective of the present invention was to further purify and characterize the H. pylori blood group antigen binding (BAB) adhesin to make possible the development of methods and materials for specific and selective diagnosing and treatment of H. pylori induced infections and related diseases and the development of said methods and materials. A further and equally important objective was to determine the DNA sequences of the genes involved in the expression of this protein. These objectives were fulfilled through the protein, the DNA and the methods and materials specified herein. The DNA sequences are attached as SEQ ID NOS: 1 and 2, disclosing the babA and babB sequences, respectively. The full protein sequences are disclosed in SEQ ID NOS: 3 and 4.